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Sfem 2 medium

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SFEM II medium is a serum-free, protein-free, and chemically-defined culture medium designed for the ex vivo expansion and maintenance of hematopoietic stem and progenitor cells.

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Lab products found in correlation

Stem cell factor (scf), by Thermo Fisher Scientific (3 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions) Human cord blood cd34 positive selection kit 2, by STEMCELL (2 mentions) Clinmacs cd34 microbead kit, by Miltenyi Biotec (2 mentions) Um171, by STEMCELL (2 mentions) Dmpge2, by Cayman Chemical (2 mentions) Cd34 expansion supplement, by STEMCELL (2 mentions) Poloxamer 407, by BASF (2 mentions) Erythroid expansion supplement, by STEMCELL (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Hcd45 microbeads, by Miltenyi Biotec (1 mentions) Recombinant il 3, by R&D Systems (1 mentions) Recombinant il 7, by R&D Systems (1 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Interleukin il 3, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions) Antibody serum, by Atlanta Biologicals (1 mentions) Heparin, by Merck Group (1 mentions) Erythropoietin, by Thermo Fisher Scientific (1 mentions) Insulin, by Merck Group (1 mentions)

15 protocols using sfem 2 medium

1

Culturing Primary Human ALL Cells with MSCs

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Primary human ALL cells were obtained after informed consent at West Virginia University Cancer Center; Cincinnati Children’s Hospital Medical Center Respiration Core; and Pittsburgh Biospecimen Core under the approved Institutional Review Broad (IRB) protocols: #1310105737; STUDY19030357 and # 2011-3023. Primary human ALL cells were cultured on hTERT-immortalized primary BM mesenchymal stromal cells (MSCs)39 ,40 (link). MSCs were seeded at a density of 104 cells/cm2 in MSC medium 48 h prior to adding to ALL. ALL cells were seeded onto MSC at a density of 2 × 106 cells/ml in SFEM II medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 20% fetal calf serum (GIBCO, Life Technologies), 20 ng/ml recombinant IL-3 (R&D Systems, Abingdon, UK) and 10 ng/ml recombinant IL-7 (R&D Systems, Minneapolis, MN). ALL cells were harvested every 7 days. Non-adherent cells present in supernatant medium were washed with PBS and passed through a 15 μm filter (pluriSelect Life Science, Leipzig, Germany). ALL cells were separated from MSCs by magnetic cell separation using hCD45 microbeads (Miltenyi Biotec, Auburn CA). Viable ALL cells were counted by Trypan blue exclusion, re-suspended in fresh ALL medium, and seeded onto fresh MSC.
Wu L., Chatla S., Lin Q., Chowdhury F.A., Geldenhuys W, & Du W. (2021). Quinacrine-CASIN combination overcomes chemoresistance in human acute lymphoid leukemia. Nature Communications, 12, 6936.
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2

Isolation and Culture of Primary BCR-ABL+ ALL Cells

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Primary BCR-ABL+ ALL cells were obtained with written informed consent and in accordance with the declaration of Helsinki. Cells were isolated via ficoll density gradient and subsequently stored in liquid nitrogen. For co-culture experiments, cells were thawed in SFEM II medium (Stemcell Technologies, Cambridge, UK) supplemented with 20% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S) (Gibco, Grand Island, NY, USA), 20 ng/mL interleukin (IL)-3 and 10 ng/mL IL-7 (both Peprotech, Hamburg, Germany).
Kirchhoff H., Ricke-Hoch M., Wohlan K., Pietzsch S., Karsli Ü., Erschow S., Zweigerdt R., Ganser A., Eder M., Scherr M, & Hilfiker-Kleiner D. (2022). Chemotherapy-Free Targeted Anti-BCR-ABL+ Acute Lymphoblastic Leukemia Therapy May Benefit the Heart. Cancers, 14(4), 983.
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3

Hematopoietic Stem Cell Differentiation

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Following targeting, HSPCs derived from healthy patients or those with SCD or β-thalassemia were cultured for 14–16 d at 37 °C and 5% CO2 in SFEM II medium (STEMCELL Technologies) as previously described34 (link),35 (link). SFEM II base medium was supplemented with 100 U ml−1 penicillin/streptomycin, 10 ng ml−1 SCF, 1 ng ml−1 IL-3 (PeproTech), 3 U ml−1 erythropoietin (eBiosciences), 200 μg ml−1 transferrin (Sigma-Aldrich), 3% antibody serum (heat-inactivated; Atlanta Biologicals), 2% human plasma (umbilical cord blood), 10 μg ml−1 insulin (Sigma-Aldrich) and 3 U ml−1 heparin (Sigma-Aldrich). In the first phase, at days 0–7 (day 0 being 2 d post targeting) of differentiation, cells were cultured at 1 × 105 ml−1. In the second phase (days 7–10), cells were maintained at 1 × 105 ml−1 and IL-3 was removed from the culture. In the third phase (days 11–16), cells were cultured at 1 × 106 ml−1 and transferrin was increased to 1 mg ml−1 within the culture medium.
Cromer M.K., Camarena J., Martin R.M., Lesch B.J., Vakulskas C.A., Bode N.M., Kurgan G., Collingwood M.A., Rettig G.R., Behlke M.A., Lemgart V.T., Zhang Y., Goyal A., Zhao F., Ponce E., Srifa W., Bak R.O., Uchida N., Majeti R., Sheehan V.A., Tisdale J.F., Dever D.P, & Porteus M.H. (2021). Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nature medicine, 27(4), 677-687.
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4

CRISPR-Mediated Reprogramming of HSPCs

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sgRNAs directed at MyD88, IFNAR1, and CCR5 were synthesized by Synthego or TriLink Technologies with the three terminal nucleotides in both ends chemically modified with 2′-O-methyl-3′-phosphorothioate35 (link). Thawed CD34+ HSPC were initially pre-cultured at low density (105 cells/mL) for 3 days in CD34+ HSPC medium (SFEM II medium (STEMCELL Technologies) supplemented with 20 units/mL penicillin, 20 mg/mL streptomycin, Flt3-L (100 ng/mL), SCF (100 ng/mL), TPO (100 ng/mL), IL-6 (100 ng/mL), SR1 (0.75 µM), and UM171 (35 nM)) before being nucleoporated as shown previously37 –39 (link). Ribonucleoprotein (RNP) complexes were made by incubating Cas9 protein (Integrated DNA Technologies) with sgRNA at a molar ratio of 1:2.5 at 25 °C for 10 min prior to nucleoporation. Nucleoporation was performed using the Lonza 4D-NucleofectorTM System (program DZ100). After a 3-day recovery phase in CD34+ HSPC expansion medium, HSPC medium was changed to the medium promoting pDC differentiation as previously described.
Laustsen A., Bak R.O., Krapp C., Kjær L., Egedahl J.H., Petersen C.C., Pillai S., Tang H.Q., Uldbjerg N., Porteus M., Roan N.R., Nyegaard M., Denton P.W, & Jakobsen M.R. (2018). Interferon priming is essential for human CD34+ cell-derived plasmacytoid dendritic cell maturation and function. Nature Communications, 9, 3525.
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5

CRISPR-Cas9 Editing of NHP CCR5 Locus

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Chemically modified sgRNAs targeting the NHP CCR5 locus with 2′-O-methyl and 3′-phosphorothioate modifications at the first three 5′- and 3′-terminal RNA residues were custom-ordered from Synthego (Redwood City, CA). Lyophilized sgRNAs were resuspended in nuclease-free water at a concentration of 100 pmol/μL and stored as frozen aliquots at −80°C. TrueCut Cas9 Protein v2 (5 μg/μL) was obtained from Thermo Fisher Scientific (Waltham, MA). Prior to editing, CD34+ cells were cultured for 24 h in serum-free SFEM II medium (STEMCELL) as described above. CRISPR/Cas9 RNP complexes were formed by mixing 180 pmol of Cas9 protein with 540 pmol of sgRNA and incubated at room temperature for 10 min. RNP complexes were added to 3 million CD34+ HSPCs, and electroporation was conducted using the BTX ECM830 as described above. Cells were plated in fresh medium and recovered overnight in a 37°C, 5% CO2 incubator, followed by harvest 1, 2, and 5 days post electroporation for analysis of gene editing.
Peterson C.W., Venkataraman R., Reddy S.S., Pande D., Enstrom M.R., Radtke S., Humbert O, & Kiem H.P. (2021). Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells. Molecular Therapy. Methods & Clinical Development, 24, 30-39.
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6

Generating iPSCs from Erythroblasts

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Peripheral blood mononuclear cells were isolated from blood of healthy donors (n = 5). Erythroblasts were expanded in SFEM II medium (StemCell Technologies) containing human erythropoietin (EPO), holo-transferrin, stem cell factor (SCF) (R&D Systems), interleukin (IL)-3, and insulin-like growth factor-1 (PeproTech), as described.29 (link) Expanded erythroblasts were reprogrammed to iPSC using three reprogramming plasmids (MOS, MMK and MBX containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes) (Addgene), as described.29 (link) iPSC were expanded in E8 medium (StemCell Technologies) containing ROCK inhibitor Y27632 (1:1000; Sigma), and immunophenotyped by flow cytometry, or stained by Giemsa banding for karyotype analyses.30 The use of human peripheral blood was approved by the Institutional Review Board of Emory University.
Deng J., Lancelot M., Jajosky R., Deng Q., Deeb K., Saakadze N., Gao Y., Jaye D., Liu S., Stowell S.R., Cheng L, & Roback J.D. (2021). Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice. American journal of hematology, 97(2), 194-202.
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7

Notch Ligand-Driven Expansion of CD34+ Cells

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CB samples were processed with red blood cell lysis buffer and enriched for CD34+ cells using CliniMACS CD34 MicroBeads (Miltenyi Biotec, 130-017-501). CD34+ CB cells were then seeded onto plates coated with RetroNectin (5 μg/mL; Takara, T100A) plus Notch ligand Delta1 (2.5 μg/mL; ref. 39 (link)) overnight in SFEM II medium (StemCell Technologies, 09650FH) containing 50 ng/mL stem cell factor (SCF; StemCell Technologies, 78062), 50 ng/mL thrombopoietin (TPO; StemCell Technologies, 78210), and 50 ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L; StemCell Technologies, 78009). Cells were transduced the following day with the C/G construct at an MOI of 200 or GFP control construct at MOI of 50. Transduced cells were grown on Notch ligand at 37°C in 5% CO2 for 6 days and then sorted for GFP+ cells. Sorted GFP+ cells were either transplanted into NSG-SGM3 mice at 200,000 cells per mouse or placed in EC coculture or MC (see ref. 23 (link) and below) for long-term culture at 75,000 cells per well in a 6-well plate. In subsequent experiments using a CD34+ CB sample from another donor (CB 2, see Supplemental Figure 4), transduced cells were grown on Notch ligand for 2 days prior to placement in EC coculture or MC plating at 100,000 cells per well of a 12-well plate.
Le Q., Hadland B., Smith J.L., Leonti A., Huang B.J., Ries R., Hylkema T.A., Castro S., Tang T.T., McKay C.N., Perkins L., Pardo L., Sarthy J., Beckman A.K., Williams R., Idemmili R., Furlan S., Ishida T., Call L., Srivastava S., Loeb A.M., Milano F., Imren S., Morris S.M., Pakiam F., Olson J.M., Loken M.R., Brodersen L., Riddell S.R., Tarlock K., Bernstein I.D., Loeb K.R, & Meshinchi S. (2022). CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target. The Journal of Clinical Investigation, 132(22), e157101.
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8

Isolation and Cryopreservation of CD34+ Cells

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Mobilized peripheral blood and leukapheresis product were anonymously collected from donors undergoing stem cell mobilization at the Massachusetts General Hospital. Mononuclear cells were purified via Histopaque-1077 gradient (Sigma–Aldrich; 10771). CD34+ cells were isolated via positive magnetic bead selection using a CD34 MicroBead Kit (Miltenyi Biotec; 130-046-702) and LS columns (Miltenyi Biotec; 130-042-401), according to the manufacturer’s recommendations. CD34+ purity routinely exceeded 85%, as assessed by flow cytometry. Aliquots of 3–5 × 105 cells were frozen in SFEM II medium (STEMCELL Technologies; 09655) + 10% DMSO (Sigma; 41640) + 20% fetal bovine serum (FBS; Gibco; 10082-147) using the CoolCell LX (Corning) at −80 °C, then transferred to liquid nitrogen cryogenic storage (VWR; CryoPro). Upon thawing, CD34+ cell viability was >90%, as assessed by trypan blue (Lonza; 17-942E).
Chou D.B., Furlong B.A., Posey R.R., Kyprianou C., O’Sullivan L.R., David R., Randle S.J., Polanska U.M., Travers J., Urosevic J., Hutchinson J.N., Che J., Howley A.M., Hasserjian R.P., Prantil-Baun R, & Ingber D.E. (2022). Differential ABC transporter expression during hematopoiesis contributes to neutrophil-biased toxicity of Aurora kinase inhibitors. Nature Communications, 13, 6021.
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9

Coculture of Patient-Derived AML Cells with Mesenchymal Stem Cells

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The primary AML samples obtained with written informed consent were provided by the Princess Maxima Center for Pediatric Oncology or St. Jude Children’s Research Hospital. Five UBTF-TD samples with co-occurring FLT3-ITD and WT1 mutations and 1 sample with RUNX1::RUNX1T1 fusion were used for in vitro coculture experiments. Patient-derived AML cells were cultured in a serum-free condition on healthy bone marrow–derived mesenchymal stem cells (MSCs). MSCs were seeded at a density of 7500 cells/cm2 in Dulbecco modified Eagle medium, low glucose, GlutaMAX(TM), pyruvate (Gibco BRL, #21885) medium supplemented with 20% FBS, 8 ng/mL fibroblast growth factor-2 (PeproTech, London, United Kingdom) and 100 U/ml penicillin/streptomycin (Gibco BRL, Life Technologies, Breda, The Netherlands), and cultivated in a 37°C, 5% CO2 incubator until reaching 70% confluence. Primary AML cells were thawed and seeded over an MSC layer at a density of 5×105 cells/mL in SFEMII medium (STEMCELL Technologies, Cologne, Germany) supplemented with 100 U/mL penicillin/streptomycin (Gibco BRL), 10 ng/mL FLT3 ligand, 10 ng/mL GM-CSF, 10 ng/mL IL-3, 150 ng/mL SCF, 100 ng/mL TPO (all from PeproTech), 750 nM SR1 (Biogeme, Lausanne, Switzerland), and 1.35 μM UM729 (STEMCELL Technologies). Cocultures were maintained at 37°C with 5% CO2 and expanded by adjusting AML cell numbers to 5 × 105 cells/mL every 4 days.
Barajas J.M., Rasouli M., Umeda M., Hiltenbrand R., Abdelhamed S., Mohnani R., Arthur B., Westover T., Thomas ME I.I.I., Ashtiani M., Janke L.J., Xu B., Chang T.C., Rosikiewicz W., Xiong E., Rolle C., Low J., Krishan R., Song G., Walsh M.P., Ma J., Rubnitz J.E., Iacobucci I., Chen T., Krippner-Heidenreich A., Zwaan C.M., Heidenreich O, & Klco J.M. (2023). Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors. Blood, 143(7), 619-630.
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10

Expansion and Drug Treatment of CD34+ Cells

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CD34+ cells were seeded into individual wells of a 96 well tissue-culture treated flat bottom polystyrene plate (1 × 104 cells/well in 200 µL) in SFEM II medium (STEMCELL Technologies; 09655) supplemented with 10% FBS (Gibco; 10082-147), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; 15140-122), 12.5 μg/ml aprotinin (Sigma–Aldrich; A3428), 20 ng/ml EPO (PeproTech; 100-64), 1 ng/ml G-CSF (PeproTech; 300-23), 100 ng/ml Flt3-L (PeproTech; 300-19), 100 ng/ml TPO (PeproTech; 300-18), 50 ng/ml SCF (PeproTech; 300-07) and select EGM-2 BulletKit (Lonza; CC-4176) components (hFGF-B, VEGF, R3-IGF-1, hEGF, ascorbic acid and heparin), according to the manufacturer’s instructions. Medium was changed on days 2 and 4 and every day afterwards. Cells were split 2x on both day 7 and day 10 of culture.
AZD2811 was obtained from AstraZeneca while Danusertib and Tozasertib were purchased from Selleckchem. Drugs were reconstituted in DMSO, stored frozen at −20 C, and then diluted into the above cell culture medium for use. To start drug treatment on day 10, cells were harvested into V-bottom polypropylene plates and centrifuged at room temperature for 5 min at 300xg. The supernatant was then aspirated and the cells were resuspended in medium containing drug or DMSO and transferred back to 96 well tissue-culture treated flat bottom polystyrene plates.
Chou D.B., Furlong B.A., Posey R.R., Kyprianou C., O’Sullivan L.R., David R., Randle S.J., Polanska U.M., Travers J., Urosevic J., Hutchinson J.N., Che J., Howley A.M., Hasserjian R.P., Prantil-Baun R, & Ingber D.E. (2022). Differential ABC transporter expression during hematopoiesis contributes to neutrophil-biased toxicity of Aurora kinase inhibitors. Nature Communications, 13, 6021.
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